Methods for sequencing polynucleotides

ABSTRACT

Provided herein is a method for sequencing a polynucleotide molecules. The method includes the steps of providing a plurality of polynucleotide molecules attached to a surface, wherein a first portion of each polynucleotide molecule is attached to a first location of the surface and a second portion of each polynucleotide molecule is attached to a second location of the surface, the relative proximity of the first and second locations being correlated with the probability that the first and second portions are paired, separating the first and second portions of the polynucleotide molecules on the surface, determining the sequences of the first and second portions of the polynucleotide molecules and comparing the relative proximities and the sequences to determine which first and second portions are paired and to determine the sequence of the target polynucleotide molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/436525 filed Jun. 10, 2019, now U.S. Pat. No. 11,279,975 which is acontinuation of U.S. application Ser. No. 14/683,580, filed on Apr. 10,2015, now U.S. Pat. No. 10,329,613, which is a continuation of U.S.application Ser. No. 13/219,422, filed on Aug. 26, 2011, now U.S. Pat.No. 9,029,103, which claims priority to U.S. Prov. App. No. 61/377,732,filed Aug. 27, 2010, which are each incorporated by reference herein inits entirety.

BACKGROUND

The technique of paired-end (PE) or pairwise sequencing is generallyknown. Paired-end sequencing allows the determination of two or morereads of sequence from two places on a single polynucleotide duplex. Theadvantage of the paired-end approach is that there is significantly moreinformation to be gained from sequencing two stretches from a singletemplate than from sequencing each of two independent templates in arandom fashion. With the use of appropriate software tools for theassembly of sequence information it is possible to make use of theknowledge that the paired-end sequences are not completely random, butare known to occur on a single duplex, and are therefore linked orpaired in the genome. This information has been shown to greatly aid theassembly of whole genome sequences into a consensus sequence.

SUMMARY

Provided herein is a method for sequencing a plurality of polynucleotidemolecules. The method includes the steps of providing a plurality ofpolynucleotide molecules attached to a surface, wherein a first portionof each polynucleotide molecule is attached to a first location of thesurface and a second portion of each polynucleotide molecule is attachedto a second location of the surface, the relative proximity of the firstand second locations being correlated with the probability that thefirst and second portions are paired, separating the first and secondportions of the polynucleotide molecules on the surface, determining thesequences of the first and second portions of the polynucleotidemolecules, and comparing the relative proximities and the sequences todetermine which first and second portions are paired and to determinethe sequence of the target polynucleotide molecules.

Also provided is a method of sequencing including the steps of providinga plurality of polynucleotide molecules, each polynucleotide moleculecomprising a first and second portion of the target polynucleotidemolecule, whereby the first and second portions are paired, attachingthe plurality of polynucleotide molecules to a surface, wherein thefirst portion of each polynucleotide molecule is attached to a firstlocation of the surface and the second portion of each polynucleotidemolecule is attached to a second location of the surface, the relativeproximity of the first and second locations being correlated with theprobability that the first and second portions are paired, separatingthe first and second portions of the polynucleotide molecules on thesurface, determining the sequences of the first and second portions ofthe polynucleotide molecules, comparing the relative proximities of thefirst portions and the second portions to determine which first andsecond portions are paired, and using sequences of the paired first andsecond portions to determine the sequence of the plurality of targetpolynucleotide molecules.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and drawings, and fromthe claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic showing an exemplary method described hereinusing double stranded templates. In (1), genomic DNA is fragmented intodouble-stranded fragments followed by ligation of SBS3′ adapters ontothe 3′ ends of the fragments. In (2), each 3′ end of the ligatedfragments is attached to a solid surface by hybridizing to an unblockedP5-SBS3 oligo. The solid surface also contains blocked P5 and blocked P7oligos.

FIG. 1B is a schematic showing an exemplary method described hereinusing double stranded templates. In (3), each strand of thedouble-stranded fragment is extended in the presence of a modifiednucleotide (uracil is shown as an example). In (4), the extendedoligonucleotides are cleaved leaving a reversible block onto the 3′ end.In (5), P5-SBS3 oligos are blocked.

FIG. 1C is a schematic showing an exemplary method described hereinusing double stranded templates. In (6), the block on the extendedoligonucleotides is cleaved. In (7), a P7′-SBS8′ adapter is ligated tothe ends of the extended oligonucleotides. In (8), the block on the P5and P7 oligos is removed.

FIG. 1D is a schematic showing an exemplary method described hereinusing double stranded templates. In (9), amplification is performed togenerate clusters of nucleic acid sequences.

FIG. 2 is a schematic showing an exemplary method of direct sequencingof single double-stranded molecules. In (1), genomic DNA is fragmentedinto double-stranded fragments followed by ligation of P5′ adapters ontothe 3′ ends of the fragments. In (2), each 3′ end of the ligatedfragments is attached to a solid surface by hybridizing to P5 oligos. In(3), the P5 oligos on the surface are used as sequencing primers tosequence the two ends of the DNA fragments.

FIG. 3A is a schematic showing an exemplary method of single moleculesequencing after ligation and extension of single stranded moleculesusing a single primer immobilized to a solid surface. In (1), genomicDNA is fragmented into double-stranded fragments followed by ligation ofP5′ adapters onto the 3′ ends of the fragments. The double-strandedfragments with adapters are then denatured to produce single strandedfragments. In (2), the 5′ end of the single-stranded fragment is ligatedonto the end of a P5 oligo comprising a modified nucleotide. In (3), the3′ end of the single-stranded fragment is hybridized to a P5 oligo.

FIG. 3B is a schematic showing an exemplary method of single moleculesequencing after ligation and extension of single stranded moleculesusing a single primer immobilized to a solid surface. In (4), the P5oligo hybridized to the 3′ end of the single-stranded fragment isextended to form a double-stranded fragment. In (5), the P5 oligo isthen cleaved at the modified nucleotide. In (6), the ends of thedouble-stranded fragment are sequenced using the P5 oligos as sequencingprimers.

FIG. 4A is a schematic showing an exemplary sequencing method describedherein using transposons. In (1), genomic DNA is fragmented intodouble-stranded fragments followed by ligation of P5′ adapters onto 3′ends of the fragments. In (2), the 3′ ends of the fragments arehybridized to P5 oligos on a solid surface. In (3), the P5 oligos arepartially extended using the double-stranded fragment as a template togenerate two extended fragments.

FIG. 4B is a schematic showing an exemplary sequencing method describedherein using transposons. In (4), transposon insertion produces twoextended fragments with additional nucleic acid sequences (e.g.,primers, adapters and/or indexing tags). The fragments can then besequenced or amplified to produce clusters for sequencing.

FIG. 5 is a schematic showing another exemplary sequencing methoddescribed herein using transposons. In (1), genomic DNA is fragmentedinto double-stranded fragments followed by ligation of P5′ adapters onto3′ ends of the fragments. In (2), the 3′ ends are hybridized to P5oligos on the solid surface followed by ligation of the 5′ ends of thefragments to the P5 oligos. In (3), transposon insertion produces twosingle-stranded fragments representing the ends of the double-strandedfragment. The single-stranded fragments can then be sequenced oramplified to produce clusters for sequencing.

FIG. 6A is a schematic showing another exemplary sequencing methodprovided herein using a patterned surface. In (1) genomic DNA isfragmented into double stranded fragments followed by ligation of twodifferent adaptors onto the 3′ ends of the fragments, in this example aP5′ adaptor on one end and a P6′ adaptor on the other end. In (2), apatterned solid surface containing two types of patches is provided, onetype of patch containing immobilized P5 and P7 oligos (“a P5 patch”) andthe other type of patch containing immobilized P6 and P7 oligos (“a P6patch”). The 3′ ends of the fragments are hybridized such that one endof the fragment will hybridize to a P5 patch and a P6 patch.

FIG. 6B is a schematic showing another exemplary sequencing methodprovided herein using a patterned surface. In (4), the P5 and P6 oligosare partially extended using the double-stranded fragment as a templateto generate two extended fragments. In (5), transposon insertionproduces two extended fragments with additional nucleic acid sequences(e.g., primers, adapters and/or indexing tags), in this case a P7′primer sequence. The fragments can then be sequenced or amplified toproduce clusters for sequencing.

FIG. 7 is a schematic showing an exemplary method described herein.Double stranded fragments containing portions A and B to be sequencedare hybridized to oligos on a solid surface. The oligos are extendedusing the double stranded fragments as template to produce immobilizedsingle stranded nucleic acid molecules. The immobilized single strandednucleic acid molecules are amplified to produce clusters. The distancebetween clusters is denoted as ‘d’ and is correlated with the length ofthe double stranded fragment. The clusters are sequenced wherein thesequence of portion A is determined in one cluster and the sequence ofportion B is determined in another cluster.

FIG. 8 is a schematic showing a sequencing method wherein the doublestranded fragments containing portions A and B are separated beforehybridization to a solid surface. In this schematic each strand willgenerate a separate cluster. The distance separating the clusters isdenoted by ‘d.’ However, in this case, ‘d’ will be completely random andwill not be correlated to the length of the double stranded fragment.

FIG. 9A is a graph showing the sequencing of an E. coli library with anaverage fragment size of 150 base pairs. According to the schematicshown in FIG. 7, there should be a correlation between the length of thefragment and the distance separating paired clusters of portions A andB. FIG. 9A shows that, when the double stranded fragments are separatedprior to hybridization to the surface, the distance between pairs ofclusters is random.

FIG. 9B is a graph showing the sequencing of an E. coli library with anaverage fragment size of 150 base pairs. In contrast, FIG. 9B showsthat, when portions A and B are separated after the double strandedfragments are hybridized to the surface, the distance between pairedclusters of portions A and B is non-random and corresponds to the sizeof the double stranded fragments.

DETAILED DESCRIPTION

General methods of paired-end sequencing have been described, forexample, in Bentley et al., Nature, 456:53-58 (2008); WO 07/010252; WO07/091077; WO 08/041002 and WO 09/032167, which are incorporated byreference herein in their entireties. Provided herein are methods forobtaining paired end (PE) information from a single read. This isachieved by using DNA fragments of sufficient length such that the twoends of a fragment generate a pair of clusters that tend to be in closeproximity. For example, in the provided methods, the DNA molecules aresufficiently long such that the two ends of the DNA molecule arecaptured on a surface in such a way that they are sufficiently separatedfrom one another so that they can be differentiated, e.g., by theformation of two optically distinct clusters.

The methods described herein advantageously permit simultaneousdetermination of sequence information for an entire DNA fragment or bothends of a DNA fragment. This will accelerate the rate at whichpolynucleotide sequence information can be obtained and improve thereliability of such sequence information, including the ability toassign empirically determined nucleotide sequences with greaterconfidence to specific locations within a larger organizationalframework, such as a gene, chromosomal region, chromosome or genome.

Provided herein is a method for sequencing a plurality of polynucleotidemolecules. The method includes attaching a plurality of polynucleotidemolecules to a surface, wherein each polynucleotide molecule comprises afirst and second portion and wherein each polynucleotide molecule isattached under conditions wherein the first portion is attached to afirst location of the surface and the second portion is attached to asecond location of the surface, separating the first and secondportions, sequencing the first and second portions, and comparing thesequences and locations or relative proximities of the first and secondportions to determine the sequence of the plurality of polynucleotidemolecules.

Thus, provided herein is a method for sequencing a plurality ofpolynucleotide molecules including the steps of providing a plurality ofpolynucleotide molecules attached to a surface, wherein a first portionof each polynucleotide molecule is attached to a first location of thesurface and a second portion of each polynucleotide molecule is attachedto a second location of the surface, the relative proximity of the firstand second locations being correlated with the probability that thefirst and second portions are paired. The method also includes the stepsof separating the first and second portions of the polynucleotidemolecules on the surface, determining the sequences of the first andsecond portions of the polynucleotide molecules, and comparing therelative proximities and the sequences to determine which first andsecond portions are paired and to determine the sequence of the targetpolynucleotide molecules.

Also provided is a method of sequencing including the steps of providinga plurality of polynucleotide molecules, each polynucleotide moleculecomprising a first and second portion of the target polynucleotidemolecule, whereby the first and second portions are paired, attachingthe plurality of polynucleotide molecules to a surface, wherein thefirst portion of each polynucleotide molecule is attached to a firstlocation of the surface and the second portion of each polynucleotidemolecule is attached to a second location of the surface, the relativeproximity of the first and second locations being correlated with theprobability that the first and second portions are paired, separatingthe first and second portions of the polynucleotide molecules on thesurface, determining the sequences of the first and second portions ofthe polynucleotide molecules, comparing the relative proximities of thefirst portions and the second portions to determine which first andsecond portions are paired, and using sequences of the paired first andsecond portions to determine the sequence of the plurality of targetpolynucleotide molecules.

In another embodiment, provided is a method of sequencing a targetpolynucleotide molecule including the steps of providing a plurality ofpolynucleotide molecules, each polynucleotide molecule comprising afirst portion and a second portion of the target polynucleotidemolecule, wherein the first and second portions are paired, attachingthe plurality of polynucleotide molecules to a surface, wherein thefirst portion of each polynucleotide molecule is attached to a firstlocation of the surface and the second portion of each polynucleotidemolecule is attached to a second location of the surface, the proximityof the first and second locations being correlated with the probabilitythat the first and second portions are paired, separating the first andsecond portions of the polynucleotide molecules thereby unpairing thefirst and second portions, sequencing the first and second portions ofthe polynucleotide molecules, comparing, at a first location, thesequence of a first or second portion to the sequences of first andsecond portions at locations in proximity to the first location, andrepeating the comparing step to determine which first and secondportions are paired and to determine the sequence of the targetpolynucleotide molecule.

As used throughout, the terms “paired” and “linked” when in reference toportions of polynucleotide molecules means that the portions occur on asingle polynucleotide molecule (e.g., the same gene, chromosome, etc.)and are, thus, linked or paired in the genome. By way of example, afterfragmentation of a plurality of polynucleotide molecules, the fragmentswill contain paired portions, i.e., portions that come from the samepolynucleotide molecule. The paired portions at the two ends of thefragments are known to be located on the same polynucleotide molecule(e.g., gene, chromosome, and the like) approximately the length of thefragment apart. This information facilitates, for example, the assemblyof a single polynucleotide sequence, a plurality of polynucleotidesequences, and the like.

In the methods described throughout, the first and second portions arepreferably separated after being attached to the surface. Optionally,the first and second portions are noncontiguous portions. As usedthroughout, the term “noncontiguous” means that the polynucleotidemolecule comprises two or more sequences that belong to the sametemplate or target polynucleotide molecule, wherein the sequences arenot adjacent on the polynucleotide molecule. For example, apolynucleotide molecule contains two noncontiguous portions that comefrom the same chromosome, but the noncontiguous portions are not locatedadjacent to one another on the polynucleotide molecule or on thechromosome. Alternatively, the first and second portions are locatedadjacent to one another on the sample polynucleotide molecule orchromosome.

In the methods described herein, the step of comparing the sequences andlocations can include using the knowledge that the portions are likelyto be located in locations closer together than locations containingunlinked sequences (i.e., sequences not on the same template orpolynucleotide molecule that was attached to the surface, e.g., the samefragment). Optionally, the step of comparing the sequences and locationsincludes using an algorithm that takes into account the first and secondportions in relative proximities are more likely to be paired orcomprise sequences from the same polynucleotide molecule (e.g., from thesame chromosome) or fragment thereof. For example, the distance betweenthe first and second portions on the surface is positively correlatedwith the probability that the first and second portions are from thesame polynucleotide molecule.

Optionally, the step of comparing the sequences and locations includesthe use of indexing tags to identify locations containing linked orpaired sequences (i.e., sequences on the same template or polynucleotidemolecule, e.g., the same fragment). Thus, the step of comparing thesequences and locations of the portions may include the use of indexingtags to identify portions containing linked sequences (i.e., sequenceson the same template or polynucleotide molecule, e.g., the samefragment). For example, a polynucleotide molecule comprising first andsecond portions may contain the same or a different indexing tag on boththe first and second portions. This can be accomplished, for example, byligating indexing tags to the ends of a polynucleotide moleculecomprising the first and second portions. Optionally, the first andsecond portions retain the tag after separation of the portions.

The step of comparing the sequences and relative proximities (orlocations) of the portions may include use of the knowledge that thesize of a cluster (generation from a single portion) is positivelycorrelated with the size of the nucleic acid molecule used to generatethe cluster. For example, a nucleic acid molecule of 100 nucleotides inlength will generate a cluster of a size larger than a nucleic acidmolecule of 50 nucleotides in length. Thus, if the first and secondportions of a polynucleotide molecule differ in length, upon separation,the first and second portions will generate clusters of different sizesproportional to the length of the first and second portions. By way ofexample, if a polynucleotide molecule is 4000 base pairs in length andit is divided into a first portion of 500 nucleotides in length and asecond portion of 3500 nucleotides in length, the cluster comprising thefirst portion will be smaller than the cluster comprising the secondportion. This information can be exploited to identify clusters likelyto contain linked sequences (i.e., the first and second portions fromthe same target polynucleotide molecule). As described in more detailbelow, the first and second clusters on the surface may be spatiallycorrelated based on the length of the polynucleotide molecule.

By way of further example, a method for sequencing a targetpolynucleotide molecule can include the steps of attaching a pluralityof polynucleotide molecules to a surface, wherein each polynucleotidemolecule comprises a first and second portion from the targetpolynucleotide molecule and wherein each polynucleotide molecule isattached under conditions wherein the first portion is attached to afirst area of the surface and the second portion is attached to a secondarea of the surface, separating the first and second portions of thepolynucleotide molecules, sequencing the first and second portions ofthe polynucleotide molecules, comparing the sequences of the firstportions and the locations of the first areas to the locations of thesecond areas and the sequences of the second portions to determine thesequence of the target polynucleotide molecule. Optionally, the firstand second portions are noncontiguous portions. Optionally, the targetpolynucleotide molecule is fragmented and the fragments are used togenerate the plurality of polynucleotide molecules, wherein eachpolynucleotide molecule comprises a first and second portion from thesame fragment. Optionally, the portions are separated by extending theattached polynucleotide molecules under conditions to incorporatecleavable sites into the extended polynucleotide molecules and cleavingthe sites of the extended oligonucleotide molecules to separate thefirst and second portions. The extension can be carried out in thepresence of one or more modified nucleotides, for example, uracil or8-oxo-guanine. Optionally, the surface comprises a plurality of firstoligos comprising a reversible block and a plurality of second,unblocked oligos to which the polynucleotide molecules attach.Optionally, after the first and second portions are separated an adapteris ligated onto the first and second portions followed by unblocking ofthe first oligos. Optionally, the adapters bind to the first oligos andthe first and second portions are amplified to produce multiple copiesof the first and second portions in the first and second areas.

By way of another example, a method for sequencing a genome includes thesteps of providing a surface comprising a plurality of clusterscomprising polynucleotide molecules, wherein each cluster comprisespolynucleotide molecules of the same sequence, determining the sequenceof the polynucleotide molecules in the clusters, comparing the sequenceof polynucleotide molecules in a first cluster to the sequence ofpolynucleotide molecules in a second cluster and comparing the locationsof the first and second clusters on the surface, and repeating thecomparing step to determine the sequence of the genome. Optionally, eachcluster is located at a known location on the surface. Optionally, thepolynucleotide molecules in the clusters are generated from one or moretarget polynucleotide molecules. The clusters can be generated by (i)attaching a plurality of polynucleotide molecules to the surface,wherein each polynucleotide molecule comprises a first and secondportion and wherein each polynucleotide molecule is attached underconditions wherein the first portion is attached to a first area of thesurface and the second portion is attached to a second area of thesurface, (ii) separating the first and second portions, and (iii)amplifying the first and second portions to produce the plurality ofclusters. The plurality of polynucleotide molecules can be produced byfragmenting one or more target polynucleotide molecules and using thefragments to generate the plurality of polynucleotide molecules, whereineach polynucleotide molecule comprises a first and second portion fromthe same fragment. Optionally, the first and second portions arenoncontiguous. As described throughout, the distance between the firstand second clusters on the surface is positively correlated with theprobability that the first and second clusters are from the same targetpolynucleotide molecule. For example, the shorter the distance betweenthe first and second clusters indicates that the first and secondclusters comprise polynucleotide molecules of sequences from the sametarget polynucleotide molecule.

As used throughout, oligonucleotides or polynucleotide molecules includedeoxyribonucleic acids (DNA), ribonucleic acids (RNA) or other form ofnucleic acid. The polynucleotide molecule can be any form of natural,synthetic or modified DNA, including, but not limited to, genomic DNA,copy DNA, complementary DNA, or recombinant DNA. Alternatively, thepolynucleotide molecule can be any form of natural, synthetic ormodified RNA, including, but not limited to mRNA, ribosomal RNA,microRNA, siRNA or small nucleolar RNA. The polynucleotide molecule canbe partially or completely in double-stranded or single-stranded form.The terms “nucleic acid,” “nucleic acid molecule,” “oligonucleotide,”and “polynucleotide” are used interchangeably throughout. The differentterms are not intended to denote any particular difference in size,sequence, or other property unless specifically indicated otherwise. Forclarity of description the terms may be used to distinguish one speciesof molecule from another when describing a particular method orcomposition that includes several molecular species.

As used throughout, the term “target polynucleotide molecule” refers tothe molecule used to generate the plurality of polynucleotide moleculesthat is attached to a surface. Target polynucleotide molecules can beany molecule to be sequenced. For example, the polynucleotide moleculecan be a plasmid, a gene, chromosomal region, chromosome or genome. Inthe context of genome or whole genome sequencing, a plurality of targetpolynucleotide molecules (e.g., a plurality of chromosomes) can be usedto generate the plurality of polynucleotide molecules.

Polynucleotide molecules or nucleic acids for use in the providedmethods may be obtained from any biological sample using known, routinemethods. Suitable biological samples include, but are not limited to, ablood sample, biopsy specimen, tissue explant, organ culture, biologicalfluid or any other tissue or cell preparation, or fraction or derivativethereof or isolated therefrom. The biological sample can be a primarycell culture or culture adapted cell line including but not limited togenetically engineered cell lines that may contain chromosomallyintegrated or episomal recombinant nucleic acid sequences, immortalizedor immortalizable cell lines, somatic cell hybrid cell lines,differentiated or differentiatable cell lines, transformed cell lines,stem cells, germ cells (e.g. sperm, oocytes), transformed cell lines andthe like. For example, polynucleotide molecules may be obtained fromprimary cells, cell lines, freshly isolated cells or tissues, frozencells or tissues, paraffin embedded cells or tissues, fixed cells ortissues, and/or laser dissected cells or tissues. Biological samples canbe obtained from any subject or biological source including, forexample, human or non-human animals, including mammals and non-mammals,vertebrates and invertebrates, and may also be any multicellularorganism or single-celled organism such as a eukaryotic (includingplants and algae) or prokaryotic organism, archaeon, microorganisms(e.g. bacteria, archaea, fungi, protists, viruses), and aquaticplankton.

The polynucleotide molecule, target polynucleotide molecule or fragmentsdescribed herein can be of any length suitable for use in the providedmethods. For example, the polynucleotide molecules or fragments can beat least 10, at least 20, at least 30, at least 40, at least 50, atleast 50, at least 100, at least 150, at least 200, at least 250, atleast 500, or at least 1000 nucleotides in length. Optionally, thepolynucleotide molecule or fragment is 150 to 4000 nucleotides inlength, 500 to 3000 nucleotides in length, or 1000 to 2000 nucleotidesin length. By way of another example, the target polynucleotidemolecules can be, for example, at least 1 kilobase in length, at least10 kilobases in length, at least 20 kilobases in length, at least 30kilobases in length, at least 40 kilobases in length, at least 50kilobases in length, at least 60 kilobases in length, at least 70kilobases in length, at least 80 kilobases in length, at least 90kilobases in length, at least 100 kilobases in length, or longer.

A plurality of polynucleotide molecules can be prepared by fragmentingone or more polynucleotide molecules and using the fragments to generatethe plurality of polynucleotide molecules comprising the first andsecond portions. Preferably, the first and second portions are from thesame fragment. In the provided methods described herein, the first andsecond portions are, optionally, located at the opposite ends of thepolynucleotide molecules (e.g., the 5′ and 3′ ends). The number ofnucleotides between the first and second portions may be substantiallythe same for each polynucleotide molecule. Optionally, the first andsecond portions are noncontiguous portions.

The plurality of polynucleotide molecules may be prepared using avariety of standard techniques available and known. Exemplary methods ofpolynucleotide molecule preparation include, but are not limited to,those described in Bentley et al., Nature 456:49-51 (2008); U.S. Pat.No. 7,115,400; and U.S. Patent Application Publication Nos.2007/0128624; 2009/0226975; 2005/0100900; 2005/0059048; 2007/0110638;and 2007/0128624, each of which is herein incorporated by reference inits entirety. For example, polynucleotide molecules are modified tocomprise one or more regions of known sequence (e.g., an adapter and/oran indexing tag) located on the 5′ and/or 3′ ends. Optionally, theadapter comprises the indexing tag. When the polynucleotide moleculescomprise known sequences on the 5′ and 3′ ends, the known sequences canbe the same or different sequences. Optionally, as described more fullybelow, a known sequence located on the 5′ and/or 3′ ends of thepolynucleotide molecules is capable of hybridizing to one or moreoligonucleotides immobilized on a surface. For example, a polynucleotidemolecule comprising a 5′ known sequence may hybridize to a firstplurality of oligonucleotides while the 3′ known sequence may hybridizeto a second plurality of oligonucleotides. Optionally, polynucleotidemolecules comprise one or more detectable labels. The one or moredetectable labels may be attached to the nucleic acid template at the 5′end, at the 3′ end, and/or at any nucleotide position within the nucleicacid template. The polynucleotide molecules for use in the providedmethods comprise the nucleic acid to be amplified and/or sequenced and,optionally, short nucleic acid sequences at the 5′ and/or 3′ end(s).

A short nucleic acid sequence that is added to the 5′ and/or 3′ end of anucleic acid can be a universal sequence. A universal sequence is aregion of nucleotide sequence that is common to, i.e., shared by, two ormore nucleic acid molecules, where the two or more nucleic acidmolecules also have regions of sequence differences. A universalsequence that may be present in different members of a plurality ofnucleic acid molecules can allow the replication or amplification ofmultiple different sequences using a single universal primer that iscomplementary to the universal sequence. Similarly, at least one, two(e.g., a pair) or more universal sequences that may be present indifferent members of a collection of nucleic acid molecules can allowthe replication or amplification of multiple different sequences usingat least one, two (e.g., a pair) or more single universal primers thatare complementary to the universal sequences. Thus, a universal primerincludes a sequence that can hybridize specifically to such a universalsequence. The target nucleic acid sequence-bearing molecules may bemodified to attach universal adapters (e.g., non-target nucleic acidsequences) to one or both ends of the different target sequences, theadapters providing sites for hybridization of universal primers. Thisapproach has the advantage that it is not necessary to design a specificpair of primers for each template to be generated, amplified, sequenced,and/or otherwise analyzed; a single pair of primers can be used foramplification of different templates provided that each template ismodified by addition of the same universal primer-binding sequences toits 5′ and 3′ ends.

The polynucleotide molecules can be modified to include any nucleic acidsequence desirable using standard, known methods. Such additionalsequences may include, for example, restriction enzyme sites, oroligonucleotide indexing tag in order to permit identification ofamplification products of a given nucleic acid sequence. As describedherein, the indexing tag can be added to a polynucleotide molecule byinclusion on an adapter or on a transposon. Optionally, the indexing tagcan be directly ligated to the ends of a polynucleotide molecule.

Optionally, the surface comprises one or more pluralities ofoligonucleotide molecules. The terms “oligonucleotides,”“oligonucleotide molecules” and “oligos” are used throughoutinterchangeably. By way of example, the surface can comprise a first,second, third, fourth, or more pluralities of oligonucleotide moleculeseach plurality having a different sequence. It will be understood thatdifferent pluralities of oligonucleotides can share a common sequence solong as there is a sequence difference between at least a portion of thedifferent pluralities. For example, as shown in FIG. 1, the two oligosidentified as P5 and P5-SBS3 share a common sequence P5, but the P5-SBS3has an additional sequence not found on the P5 oligo. Thus, a firstplurality of oligonucleotides can share a sequence with a secondplurality of oligonucleotides as long as the oligos in one pluralityhave a different sequence not found in the oligos of the otherplurality.

Once the plurality of polynucleotide molecules is prepared, one or moreof the polynucleotide molecules in the plurality of polynucleotidemolecules can be attached to a surface. The one or more of thepolynucleotide molecules can be attached to the surface under conditionswherein the first portion is attached to a first location of the surfaceand the second portion is attached to a second location of the surface.

The polynucleotide molecules can be attached to the surface byhybridization or binding to a plurality of oligos. Optionally, thepolynucleotide molecules are attached to the surface by attaching oneend of the polynucleotide molecule to the surface or to the end of theoligos (e.g., by ligation). Hybridization is accomplished, for example,by ligating an adapter to the ends of the polynucleotide molecules. Thenucleic acid sequence of the adapter can be complementary to the nucleicacid sequence of the oligo, thus, allowing the adapter to bind orhybridize to the oligos on the surface. Optionally, the polynucleotidemolecules are single or double stranded and adapters are added to the 5′and/or 3′ ends of the polynucleotide molecules. Optionally, thepolynucleotide molecules are double-stranded and adapters are ligatedonto the 3′ ends of double-stranded polynucleotide molecule. Optionally,polynucleotide molecules are used without any adapter.

By way of another example, the surface comprises a plurality of firstoligos to which the polynucleotide molecules attach and a plurality ofsecond oligos comprising a reversible block. As described above, thepolynucleotide molecules can hybridize to the first oligos through anadapter. After the first and second portions are separated, a secondadapter can be ligated onto the ends of the first and second portions.In this aspect, this part of the sample prep can take place inside theflowcell. The nucleic acid sequence of the second adapter can becomplementary to the nucleic acid sequence of the second oligo. Thesecond oligos can be unblocked and the second adapters can bind to thesecond oligos. The first and second portions can then be amplified toproduce multiple copies of the first and second portions in the firstand second locations. Thus, the first and second locations can beclusters of polynucleotide molecules comprising first and secondportions. Optionally, the first oligos, second oligos or adapterscomprise an oligonucleotide indexing tag.

By way of a third example, the surface comprises a plurality of firstoligos to which the polynucleotide molecules attach and a plurality ofsecond and third oligos comprising a reversible block. After thepolynucleotide molecules attach to the surface through hybridization tothe first oligos, the polynucleotide molecule can be extended. Ifdouble-stranded, each strand of the double-stranded polynucleotidemolecule can be extended, e.g., in the presence of a modifiednucleotide, in order to facilitate separation of the first and secondportions of the polynucleotide molecules. After the first and secondportions are separated, a second adapter can be ligated to the ends ofthe extended first and second portions followed by removal of the blocksfrom the second and third oligos. The first and second portions can thenbe amplified to produce multiple copies of the first and second portionsin discrete locations referred to herein as clusters. Optionally, thefirst oligos, second oligos, third oligos or adapters comprise anoligonucleotide indexing tag.

By way of another example, the surface is a patterned surface andcomprises a two or more types of patches. A first type of patch containsa plurality of first oligos and a plurality of second oligos. The secondtype of patch contains a plurality of the second oligos and a pluralityof third oligos. Polynucleotide molecules are hybridized such that oneend of the molecules hybridizes to a first patch and the other end ofthe molecule hybridizes to a second patch. After the first and secondportions are separated, they can then be sequenced or amplified toproduce clusters for sequencing.

A surface or support for use in the provided methods described hereinrefers to any surface or collection of surfaces to which nucleic acidscan be attached. Suitable surfaces include, but are not limited to,beads, resins, gels, wells, columns, chips, flowcells, membranes,matrices, plates or filters. For example, the surface can be latex ordextran beads, polystyrene or polypropylene surfaces, polyacrylamidegels, gold surfaces, glass surfaces, optical fibers, or silicon wafers.Optionally, the surface is three dimensional, for example, a threedimensional matrix. The surface can be any material that is amenable tolinkage to a nucleic acid.

Optionally, the surface is contained in a vessel or chamber such as aflow cell, allowing convenient movement of liquids across the surface toenable the transfer of reagents. Exemplary flow cells that can be usedin this manner are described in WO 2007/123744, which is incorporatedherein by reference in its entirety.

Optionally, the surface may comprise a layer or coating of a materialwith reactive groups permitting attachment of polynucleotides. Thepolynucleotides are then attached to the material (e.g., covalently),which is attached to the surface (e.g., noncovalently). Such a surfaceis described in WO 05/65814, which is incorporated by reference hereinin its entirety.

The term “immobilized” as used herein is intended to encompass direct orindirect attachment to a solid support via covalent or non-covalentbond(s). In particular embodiments, all that is required is that themolecules (for example, nucleic acids) remain immobilized or attached toa support under conditions in which it is intended to use the support,for example in applications requiring nucleic acid amplification and/orsequencing. For example, oligonucleotides are immobilized such that a 3′end is available for enzymatic extension and/or at least a portion ofthe sequence is capable of hybridizing to a complementary sequence.Immobilization can occur via hybridization to a surface attachedoligonucleotide, in which case the immobilized oligonucleotide orpolynucleotide may be in the 3′-5′ orientation. Alternatively,immobilization can occur by means other than base-pairing hybridization,such as the covalent attachment.

In particular embodiments, the attached polynucleotide moleculescomprise a cleavable site to separate the first and second portions. Forexample, the attached polynucleotide molecules can be extended underconditions to incorporate cleavable sites into the extendedpolynucleotide molecules and cleaving the sites of the extendedoligonucleotide molecules to separate the first and second portions.Various cleavage methods may be used in accordance with the providedmethods to cleave one or both strands of the polynucleotide molecules.Optionally, the cleavable site comprises a modified nucleotide or arestriction enzyme site. Such methods are known and include thosedescribed in U.S. Publication No. 20090118128, which is incorporated byreference herein in its entirety. For example, chemical cleavage may beused, which encompasses any method using a non-enzymatic chemicalreagent in order to promote/achieve cleavage of a polynucleotidemolecule whether in single or double stranded form. The polynucleotidemolecule can include one or more non-nucleotide chemical moieties and/ornon-natural nucleotides and/or non-natural backbone linkages in order topermit a chemical cleavage reaction at a pre-determined cleavage site.By way of example, in the provided methods, the extension is carried outin the presence of one or more types of modified nucleotides.Nucleotides for use in the provided methods include, for example,derivatives capable of being selectively cleaved in a nucleic acidstrand. Such nucleotides include, but are not limited to, uracil or8-oxo guanine. Optionally, two types of modified nucleotides can be used(e.g., uracil and 8-oxo guanine, e.g., to reduce GC bias). Thesemodified nucleotides can be modified to abasic sites by the actions ofUracil DNA glycosylase (UDG) and formamidopyrimidine ##fapy##-DNAglycosylase (FPG), respectively. The polynucleotide strand including theabasic site may then be cleaved at the abasic site by treatment withendonuclease (e.g. EndolV endonuclease, AP lyase, FPG glycosylase/APlyase, and EndoVIII glycosylase/AP lyase), heat or alkali. FPG alone canalso result in abasic site cleavage.

The provided methods may make use of non-extendable nucleotides whichact as terminators and prevent further strand elongation. Suchterminators may be permanent (e.g., dideoxyribose analogues such asddTTP or ddATP) or reversible. Reversible terminators may contain anymoiety which acts to block polymerase extension, but can subsequently bealtered to allow polymerase extension. Suitable reversible terminatormoieties include blocking groups on the nucleotide 3′ hydroxyl. There isa variety of known 3′ hydroxyl blocking moieties that are capable ofacting as reversible polymerase blocks, including the allyl,methoxymethyl, azidomethyl or O—NH2 groups. Optionally, terminatormoieties are attached to nucleotide bases at 2′ or 4′ positions.Examples of nucleotide terminators can be found in U.S. Pat. Nos.5,302,509; 7,057,026; 6,664,079; 7,541,444; and 7,544,794, the contentsof which are incorporated by reference herein in their entireties. Ifdesired, reversible terminators may be removed to allow subsequentpolymerase action on the strands, for example to synthesize full lengthstrands at the end of the amplification process.

The modified nucleotides are typically provided at a concentrationeffective to generate immobilized polynucleotide molecules of anappropriate size. Such concentrations may be determined empirically bythose of skill in the art. For example, the concentration of modifiednucleotides may be determined based on the ability of a polymerase toincorporate the modified nucleotide and/or based on the desired lengthof the fragment to be left after cleavage. For example, the modifiednucleotides are provided at a concentration ratio of 1 to 100. By way ofexample, if uracil is used, for every 100 units of dTTP supplied, 1 unitof dUTP is supplied.

Alternatively, the first and second portions can be separated throughuse of a transposon. As used herein, the term transposon refers to thenucleic acid sequence containing the transposon elements together withall of the nucleic acid sequence between the elements. Transposons maycomprise a cleavable element such as a modified nucleotide or arestriction enzyme site. Optionally, the transposon may also comprise anindexing tag. Transposons generally require only the transposase proteinand a cognate transposon. The transposase may be purified from naturalsources or it may be produced in vitro or synthesized by methods knownin the art. Transposase may be expressed in bacterial, yeast, insect ormammalian cells or produced in cell-free expression systems. Thetransposase may have a wild-type amino acid sequence or it may have amodified amino acid sequence. Modifications include mutations thataffect the activity or stability of the transposase or add functionalityto the transposase. Suitable transposon systems useful in the providedmethods include, but are not limited to, Sleeping Beauty, To12,PiggyBac, Frog Prince, Minos, and Hsmarl. Transposons also includetransposable elements found in prokaryotes such as insertion sequences(IS), transposons (Tn), or bacteriophages such as Mu and D108.Eukaryotic transposable elements include, but are not limited to: Copiaelements, TY elements, Tal and Tnt 1 transposable elements, TAP, Tam orCin transposable elements, and AC, Spm, Bs, CM, Dt, and Mutatortransposable elements. In particular embodiments, a synthetictransposable element is used that lacks a functional transposase butwhich is supplied in trans.

In particular embodiments, the polynucleotide molecules are doublestranded and the two strands of the polynucleotide molecules areseparated by denaturing the strands of the polynucleotide molecules.Each of the denatured or separated strands of the polynucleotidemolecules can then be amplified, e.g., to produce a plurality ofclusters. Clusters are described in more detail below.

In other embodiments, the first and second portions are separated byextending the primers hybridized to the double stranded polynucleotidemolecules for a period of time sufficient to produce copies of the firstand second portions. The first and second portion copies remainimmobilized while the double stranded polynucleotide molecules areremoved (e.g., by washing). The first and second portions are thenamplified, e.g., to generate clusters for sequencing or are directlysequenced.

In the provided methods, after the first and second portions areseparated, the first and second portions can be amplified prior tosequencing to produce multiple copies of the first and second portionsat the first and second locations. Nucleic acid amplification includesthe process of amplifying or increasing the numbers of a nucleic acidtemplate and/or of a complement thereof that are present, by producingone or more copies of the template and/or its complement. In theprovided methods, amplification can be carried out by a variety of knownmethods under conditions including, but not limited to, thermocyclingamplification or isothermal amplification. For example, methods forcarrying out amplification are described in U.S. Publication No.2009/0226975; WO 98/44151; WO 00/18957; WO 02/46456; WO 06/064199; andWO 07/010251; which are incorporated by reference herein in theirentireties. Briefly, in the provided methods, amplification can occur onthe surface to which the polynucleotide molecules are attached. Thistype of amplification can be referred to as solid phase amplification,which when used in reference to nucleic acids, refers to any nucleicacid amplification reaction carried out on or in association with asurface (e.g., a solid support). For example, all or a portion of theamplified products are synthesized by extension of an immobilizedprimer. Solid phase amplification reactions are analogous to standardsolution phase amplifications except that at least one of theamplification oligonucleotides is immobilized on a surface (e.g., asolid support).

Solid-phase amplification may comprise a nucleic acid amplificationreaction comprising only one species of oligonucleotide primerimmobilized to a surface. Alternatively, the surface may comprise aplurality of first and second different immobilized oligonucleotideprimer species. Solid-phase amplification may comprise a nucleic acidamplification reaction comprising one species of oligonucleotide primerimmobilized on a solid surface and a second different oligonucleotideprimer species in solution. Solid phase nucleic acid amplificationreactions generally comprise at least one of two different types ofnucleic acid amplification, interfacial and surface (or bridge)amplification. For instance, in interfacial amplification the solidsupport comprises a template polynucleotide molecule that is indirectlyimmobilized to the solid support by hybridization to an immobilizedoligonucleotide primer, the immobilized primer may be extended in thecourse of a polymerase-catalyzed, template-directed elongation reaction(e.g., primer extension) to generate an immobilized polynucleotidemolecule that remains attached to the solid support. After the extensionphase, the nucleic acids (e.g., template and its complementary product)are denatured such that the template polynucleotide molecule is releasedinto solution and made available for hybridization to anotherimmobilized oligonucleotide primer. The template polynucleotide moleculemay be made available in 1, 2, 3, 4, 5 or more rounds of primerextension or may be washed out of the reaction after 1, 2, 3, 4, 5 ormore rounds of primer extension.

In surface (or bridge) amplification, an immobilized polynucleotidemolecule hybridizes to an immobilized oligonucleotide primer. The 3′ endof the immobilized polynucleotide molecule provides the template for apolymerase-catalyzed, template-directed elongation reaction (e.g.,primer extension) extending from the immobilized oligonucleotide primer.The resulting double-stranded product “bridges” the two primers and bothstrands are covalently attached to the support. In the next cycle,following denaturation that yields a pair of single strands (theimmobilized template and the extended-primer product) immobilized to thesolid support, both immobilized strands can serve as templates for newprimer extension.

Optionally, amplification of the first and second portions results inclustered arrays of nucleic acid colonies, analogous to those describedin U.S. Pat. No. 7,115,400; U.S. Publication No. 2005/0100900; WO00/18957; and WO 98/44151, which are incorporated by reference herein intheir entireties. Thus, the first and second portions can be amplifiedto produce a plurality of clusters. Clusters and colonies are usedinterchangeably and refer to a plurality of copies of a nucleic acidsequence and/or complements thereof attached to a surface. Typically,the cluster comprises a plurality of copies of a nucleic acid sequenceand/or complements thereof, attached via their 5′ termini to thesurface. For example, as described herein, a plurality of pairs ofclusters comprising noncontiguous sequences are attached to a surface.The copies of nucleic acid sequences making up the clusters may be in asingle or double stranded form.

The clusters can have different shapes, sizes and densities depending onthe conditions used. For example, clusters can have a shape that issubstantially round, multi-sided, donut-shaped or ring-shaped. Thediameter or maximum cross section of a cluster can be from about 0.2 μmto about 6 μm, about 0.3 μm to about 4 μm, about 0.4 μm to about 3 μm,about 0.5 μm to about 2 μm, about 0.75 μm to about 1.5 μm, or anyintervening diameter. Optionally, the diameter or maximum cross sectionof a cluster can be at least about 0.5 μm, at least about 1 μm, at leastabout 1.5 μm, at least about 2 μm, at least about 2.5 μm, at least about3 μm, at least about 4 μm, at least about 5 μm, or at least about 6 μm.The diameter of a cluster may be influenced by a number of parametersincluding, but not limited to, the number of amplification cyclesperformed in producing the cluster, the length of the nucleic acidtemplate, the GC content of the nucleic acid template or the density ofprimers attached to the surface upon which clusters are formed. Thedensity of clusters can be in the range of at least about 0.1/mm², atleast about 1/mm², at least about 10/mm², at least about 100/mm², atleast about 1,000/mm², at least about 10,000/mm² to at least about100,000/mm². Optionally, the clusters have a density of, for example,100,000/mm² to 1,000,000/mm² or 1,000,000/mm² to 10,000,000/mm².Clusters may be detected, for example, using a suitable imaging means,such as, a confocal imaging device or a charge coupled device (CCD)camera. Exemplary imaging devices include, but are not limited to, thosedescribed in U.S. Pat. Nos. 7,329,860; 5,754,291; and 5,981,956; and WO2007/123744, each of which is herein incorporated by reference in itsentirety. The imaging means may be used to determine a referenceposition in a cluster or in a plurality of clusters on the surface, suchas the location, boundary, diameter, area, shape, overlap and/or centerof one or a plurality of clusters (and/or of a detectable signaloriginating therefrom). Such a reference position may be recorded,documented, annotated, converted into an interpretable signal, or thelike, to yield meaningful information. For example, the referenceposition can be interpreted by the imaging device as a signal that maybe generated from two or more adjacent, neighboring or proximalclusters, for example, in order to assist in distinguishing (i) adjacentclusters that are the products of extension from and amplification offirst and second non-contiguous regions of a common targetpolynucleotide, from (ii) unrelated clusters. The signal may, forinstance, take the form of a detectable optical signal emanating from adefined and identifiable location, such as a fluorescent signal, or maybe a detectable signal originating from any other detectable label asprovided herein. The reference position of a signal generated from twoor more clusters may be used to determine the actual physical positionon the surface of two clusters that are related by way of being thesites for simultaneous sequence reads from different portions of acommon target polynucleotide. As discussed in more detail below, thesequence information obtained from the clusters and the proximity ofclusters can in turn be used to determine the location of the sequencesfrom the clusters in a genome (or other original sequence) from whichthe clusters were derived.

Following amplification, the polynucleotide molecules can be sequenced.The sequencing is carried out by a variety of known methods, including,but not limited to, sequencing by ligation, sequencing by synthesis orsequencing by hybridization.

Sequencing by synthesis, for example, is a technique wherein nucleotidesare added successively to a free 3′ hydroxyl group, typically providedby annealing of an oligonucleotide primer (e.g., a sequencing primer),resulting in synthesis of a nucleic acid chain in the 5′ to 3′direction. These and other sequencing reactions may be conducted on theherein described surfaces bearing nucleic acid clusters. The reactionscomprise one or a plurality of sequencing steps, each step comprisingdetermining the nucleotide incorporated into a nucleic acid chain andidentifying the position of the incorporated nucleotide on the surface.The nucleotides incorporated into the nucleic acid chain may bedescribed as sequencing nucleotides and may comprise one or moredetectable labels. Suitable detectable labels, include, but are notlimited to, protons, haptens, radionucleotides, enzymes, fluorescentlabels, chemiluminescent labels, and/or chromogenic agents. One methodfor detecting fluorescently labeled nucleotides comprises using laserlight of a wavelength specific for the labeled nucleotides, or the useof other suitable sources of illumination. The fluorescence from thelabel on the nucleotide may be detected by a CCD camera or othersuitable detection means. Suitable instrumentation for recording imagesof clustered arrays is described in WO 07/123744, the contents of whichare incorporated herein by reference herein in its entirety.

Optionally, cycle sequencing is accomplished by stepwise addition ofreversible terminator nucleotides containing, for example, a cleavableor photobleachable dye label as described, for example, in U.S. Pat.Nos. 7,427,673; 7,414,116; WO 04/018497; WO 91/06678; WO 07/123744; andU.S. Pat. No. 7,057,026, the disclosures of which are incorporatedherein by reference in their entireties. The availability offluorescently-labeled terminators in which both the termination can bereversed and the fluorescent label cleaved facilitates efficient cyclicreversible termination (CRT) sequencing. Polymerases can also beco-engineered to efficiently incorporate and extend from these modifiednucleotides.

Alternatively, pyrosequencing techniques may be employed. Pyrosequencingdetects the release of inorganic pyrophosphate (PPi) as particularnucleotides are incorporated into the nascent strand (Ronaghi et al.,(1996) “Real-time DNA sequencing using detection of pyrophosphaterelease.” Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001)“Pyrosequencing sheds light on DNA sequencing.” Genome Res. 11(1), 3-11;Ronaghi, M., Uhlen, M. and Nyren, P. (1998) “A sequencing method basedon real-time pyrophosphate.” Science 281(5375), 363; U.S. Pat. Nos.6,210,891; 6,258,568; and 6,274,320, the disclosures of which areincorporated herein by reference in their entireties). Inpyrosequencing, released PPi can be detected by being immediatelyconverted to adenosine triphosphate (ATP) by ATP sulfurylase, and thelevel of ATP generated is detected via luciferase-produced photons.

Additional exemplary sequencing-by-synthesis methods that can be usedwith the methods described herein include those described in U.S. PatentPublication Nos. 2007/0166705; 2006/0188901; 2006/0240439; 2006/0281109;2005/0100900; U.S. Pat. No. 7,057,026; WO 05/065814; WO 06/064199; WO07/010251, the disclosures of which are incorporated herein by referencein their entireties.

Alternatively, sequencing by ligation techniques are used. Suchtechniques use DNA ligase to incorporate oligonucleotides and identifythe incorporation of such oligonucleotides and are described in U.S.Pat. Nos. 6,969,488; 6,172,218; and 6,306,597; the disclosures of whichare incorporated herein by reference in their entireties. Other suitablealternative techniques include, for example, fluorescent in situsequencing (FISSEQ), and Massively Parallel Signature Sequencing(VIPSS).

In traditional paired end sequencing, two sequencing reads are obtainedfrom the same polynucleotide molecule to obtain the sequences of twodifferent portions of the polynucleotide molecule. The two differentportions can be contiguous or noncontiguous. Since the differentportions are never separated, it is known that the two differentportions come from the same original or parent nucleic acid molecule(e.g., a chromosome). By way of an example, in traditional paired endsequencing on a solid surface, a cluster of polynucleotide moleculescomprising two different portions will be read twice because eachportion that is to be read is located on the same polynucleotidemolecule. In contrast, in the provided methods, the two differentportions on the ends of the polynucleotide molecules are separated(e.g., they may form separate clusters). Thus, a single read providesthe sequence information for both portions at the same time. The methodsdescribed herein provide clusters that tend to be spatially correlated.This is accomplished by using molecules sufficiently long such that thetwo ends of the molecule are captured on a surface in such a way thatthey are sufficiently separated from one another so that they can bedifferentiated optically, e.g., by the formation of two distinctclusters. The knowledge of the sequences of the polynucleotide moleculeslocated on the surface of a support (e.g., in clusters) and thelocations or relative proximities of the polynucleotide molecules can beused to determine or assemble the sequence of a target polynucleotidemolecule (e.g., a gene, chromosome, chromosomal region, genome and thelike). As described above, in the provided methods, sequences of aplurality of first and second portions are compared to the relativeproximities of the first and second locations comprising the first andsecond portions, respectively, to determine the which first and secondportions are paired and to determine the sequence of a plurality ofpolynucleotide molecules. The distance between the first and secondlocations (or first and second clusters) is correlated with theprobability that the first and second locations or clusters are from thesame target polynucleotide molecule (e.g., fragment). Optionally, thesequence and proximity of a polynucleotide molecule in a first locationor cluster is compared to the sequence and proximity of a polynucleotidemolecule in a second location or cluster. Optionally, the first andsecond portions comprise an oligonucleotide indexing tag. In otherwords, a “first” polynucleotide molecule comprising a first and secondportion will comprise the same oligonucleotide indexing tag and a“second” different polynucleotide molecule comprising a first and secondportion will comprise the same indexing tag. However, the tag for thefirst polynucleotide molecule is different from the tag for the secondpolynucleotide molecule. Since the first and second portions are locatedin different locations, the tag can be used to determine which firstportion pairs with which second portion. This information can be used todetermine the sequence of a target polynucleotide molecule (e.g., achromosome) or a plurality of target polynucleotide molecules (e.g., agenome).

Although some aspects of the methods have been described above in a wayto distinguish them from standard paired end sequencing, it will beunderstood that standard paired end sequencing techniques can be used incombination with other techniques and methods set forth herein.Specifically, paired end sequencing techniques can be used to determinethe sequence of polynucleotides within individual, respective clusterson a surface and proximity between those clusters on the surface can beused to determine the sequence of the target polynucleotide from whichthe clusters were derived. Methods for carrying out paired endsequencing that can be useful in the methods set forth herein aredescribed in the art, for example, in Bentley et al., Nature, 456:53-58(2008); WO 07/010252; WO 07/091077; WO 08/041002 and WO 09/032167, whichare incorporated by reference herein in their entireties.

The provided methods can be used for de novo sequencing orre-sequencing. In the context of re-sequencing, the sequences of thefirst and second portions are compared to a reference sequence.Information about the physical proximity of clusters on the surface of aflow cell or other substrate can be used to further confirm that twoclusters or two locations contain noncontiguous sequences that werederived from a single fragment that was seeded onto the surface asopposed to being unlinked sequences (i.e., sequences not located on thesame original or parent target polynucleotide molecule).

Once the reads from each cluster or location are obtained, algorithmsare used to re-assemble the data. For an example of paired-end readalignments and assembly see, e.g., Batzoglou et al. (2002) Genome Res.,12(1):177-189. Sequence information from each individual cluster orlocation is obtained and the clusters or locations are paired togetherbased on their proximity on the surface. In one embodiment, the shorterthe distance between clusters indicates that the clusters comprisepolynucleotide molecules of noncontiguous or contiguous sequences fromthe same parent or target polynucleotide molecule. The distance betweenclusters can be correlated with the probability that the clusters arefrom the same target polynucleotide molecule or the same fragment of thetarget polynucleotide molecule based on the length of the originalpolynucleotide molecule hybridized to the surface. Two clusters arisingfrom the ends of each strand are separated on the surface by thephysical length that is the same or less than the length of the initialfragments (approximately). In other words, the center-center distancebetween two clusters from the opposite ends of a single polynucleotidemolecule is approximately no longer than the length of thepolynucleotide molecule used to generate the clusters. Similarly, thedistance between two locations to which first and second portions ofpolynucleotide molecules are bound is no longer than the length of thepolynucleotide molecule. Nucleic acid fragment sizes for double strandedduplexes correspond to 0.34 nm per base pair. Thus, the ends of a 10 kBdouble stranded fragment should be approximately 3.4 micrometers apartor less, and a 100 kB fragment should be approximately 34 micrometersapart or less. For clusters that average around 1 micron, two clustersoriginating from the ends of long fragments will appear close to eachother on the surface.

Whether locations or clusters, using the known length of the startingfragments within a range of sizes, it is possible to work out themaximum separation possible for the two ends of each fragment. Pairingtogether all the sequences of the clusters or locations within a knownproximity on the surface gives a finite number of possible pairedsequences for a particular fragment size. This can be carried out untilthe entire sequence of the target polynucleotide molecule (e.g.,chromosome fragments) or plurality of target polynucleotide molecules(e.g., genome) is assembled. Depending on the complexity of the sample,it should be possible to discount the majority of the sequences ascoming from clearly different molecules (e.g., different chromosomefragments). For example, in the case of a human genomic DNA sample, ifthere are 6 individual sequences within a small area of surface, twofrom one chromosome fragment, two from another chromosome fragment, andtwo from a third chromosome fragment, it is straightforward to pair thesequences together. Similarly, if one of the six sequences is ambiguousfor two locations in the human genome, the identity of the other 5sequences (2 correlated pairs and one unambiguous sequence) can be usedto assist in determining where the ambiguous sequence lies in thegenome.

As discussed above, indexing tags can also be used to assist inassembling the sequence of a target polynucleotide molecule (e.g.,chromosome fragments) or plurality of target polynucleotide molecules(e.g., genome). By way of example, target polynucleotide molecule(s) canbe fragmented and adapters comprising indexing tags can be attached tothe ends of the fragment. The fragment, thus, contains first and secondportions that, when separated, have the same indexing tag.

Another embodiment that can be used in conjunction with all otherembodiments described herein includes fragmenting the targetpolynucleotide molecule with a restriction enzyme to generate non-randomends. This can be used to help determine the true ends of the fragmentbased on the knowledge that the end of the fragment will be the sequenceof the restriction enzyme site.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation may not be explicitly disclosed,each is specifically contemplated and described herein. For example, ifa method is disclosed and discussed and a number of modifications thatcan be made to the method steps are discussed, each and everycombination and permutation of the method steps, and the modificationsthat are possible are specifically contemplated unless specificallyindicated to the contrary. Likewise, any subset or combination of theseis also specifically contemplated and disclosed. This concept applies toall aspects of this disclosure. Thus, if there are a variety ofadditional steps that can be performed it is understood that each ofthese additional steps can be performed with any specific method stepsor combination of method steps of the disclosed methods, and that eachsuch combination or subset of combinations is specifically contemplatedand should be considered disclosed.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application.

EXAMPLES Example 1. Obtaining Paired-End Information from a Long DNAFragment

With reference to FIG. 1, genomic DNA is fragmented into large fragments(i.e. 100 Kb or more). After end repair, adapters are then ligated ontothe 3′ ends (e.g., adapters can be SBS3′ or SBS8′). The molecules arethen flowed inside a flowcell that has been grafted with blocked P5 andP7 oligonucleotides and also a certain amount of unblocked P5-SBS3 (orP7-SBS8). Although FIG. 1-1 refers to a P5 oligo with reversible block 1and P7 oligo with reversible block 2, it is noted that the blocks 1 and2 on P5 and P7 can be the same block or different blocks. The ends ofthe genomic DNA molecule (SBS3′ or SBS8′) will hybridize to the reversecomplement oligonucleotides present on the flowcell surface. In the nextstep, an extension with a dNTP mix that contains an optimal amount ofdUTP (or other modified nucleotide that can be cleaved to form a 3′reversible block, such as a phosphate group) is performed. The modifiednucleotide will be randomly incorporated into the growing DNA strandduring the extension step and its concentration is optimized so that DNAmolecules of an appropriate size range are generated. After extension,the modified nucleotides are cleaved and this leaves a reversible blockonto the 3′ end (i.e. phosphate). A modified nucleotide can be chosensuch that is has a 3′ reversible block and its incorporation causestermination of the extension reaction. The P5-SBS3 (or P7-SBS8)oligonucleotides that have not been hybridized and extended are thenblocked (i.e. with ddNTPs). The reversible block at the 3′ end ofgenomic DNA is now cleaved off and an adapter is ligated to the genomicDNA's 3′ ends (either SBS8′-P7′ or SBS3′-P5′). After removal of the 3′blocks from P5 and P7, amplification of the molecules and sequencing ofthe clusters is performed. The two ends of a molecule will tend tooriginate a pair of clusters that are spatially correlated. Thus, pairedend information is obtained with a single read.

Example 2. Correlation Between Fragment Size and Cluster Distance

In order to determine paired end information in a single read, thereneeds to be a correlation between fragment size and the location of thepaired portions on a surface. To determine whether there is acorrelation between fragment size and the location of paired portions ona surface, two different sequencing methods were carried out as shown inthe schematic of FIGS. 7 and 8. In FIG. 7, double stranded fragmentscontaining portions A and B to be sequenced were hybridized to asurface. In FIG. 8, the double stranded fragments containing portions Aand B were separated prior to hybridization to a surface. In both cases,each strand of the double stranded fragment was amplified to produceclusters. The clusters were sequenced wherein the sequence of portion Awas determined in one cluster and the sequence of portion B wasdetermined in another cluster. The results are shown in FIGS. 9A and 9B.FIGS. 9A and 9B are graphs showing the sequencing of an E. coli librarywith an average fragment size of 150 base pairs. FIG. 9A shows that,when the double stranded fragments are separated prior to hybridizationto the surface (as shown in FIG. 8), the distance between pairs ofclusters is random. In contrast, FIG. 9B shows that, when portions A andB are separated after the double stranded fragments are hybridized tothe surface (as shown in FIG. 7), a significant proportion of clusterpairs align against the genome at a distance that corresponds to theaverage insert size of the library used in this experiment. Thus, when asequencing method is performed wherein portions A and B are separatedafter hybridization to a surface, polynucleotide molecules can besequenced by comparing the relative proximities of the clusters ofportions A and B to determine which portions are paired and thesequences of the paired portions can be used to determine the sequenceof the polynucleotide molecules. Thus, the methods provided hereinprovide paired-end information in a single read. The provided methodssimplify paired-end sequencing while still taking advantage of theknowledge that two sequences (i.e., the two portions of the fragments)are linked or paired and, thus, known to occur on a single duplex. Thisknowledge can facilitate, for example, the assembly of whole genomesequences.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the following claims.

1. (canceled)
 2. A method for determining sequences of polynucleotidesthat potentially contain a first portion and a second portion from thesame polynucleotide, comprising: (a) providing a plurality of nucleicacid clusters attached to a surface, wherein the plurality of nucleicacid clusters is obtained by: (i) providing a plurality ofdouble-stranded polynucleotides attached to a surface, wherein a firstportion on a first strand of each of the plurality of double-strandedpolynucleotide molecules is attached to a first location of the surfaceand a second portion on a second strand of each of the plurality ofdouble-stranded polynucleotide molecules is attached to a secondlocation of the surface, wherein the first portion and the secondportion are located at the opposite ends of two complementary strands ofeach of the plurality of double-stranded polynucleotide molecules, andwherein each of the plurality of double-stranded polynucleotidemolecules has a known length; (ii) separating the first portion on thefirst strand of each of the plurality of double-stranded polynucleotidemolecules from the second portion on the second strand of each of theplurality of double-stranded polynucleotide molecules by denaturing thetwo complementary strands of each of the plurality of double-strandedpolynucleotide molecules, thereby generating a plurality of denaturedsingle polynucleotide strands, each of the plurality of denatured singlepolynucleotide strands being immobilized at the first location or thesecond location of the surface; and (iii) amplifying the first portionof each of the plurality of denatured single polynucleotide strandsimmobilized at the first location and amplifying the second portion ofeach of the plurality of denatured single polynucleotide strandsimmobilized at the second location, thereby obtaining the plurality ofnucleic acid clusters on the surface; (b) identifying the locations ofthe plurality of nucleic acid clusters on the surface using an imagingdevice, and determining the distance from one cluster of the pluralityof nucleic acid clusters to another cluster of the plurality of nucleicacid clusters on the surface; (c) determining the sequences of the firstportion and the second portion of the plurality of nucleic acidclusters; and (d) determining the sequences of the polynucleotides thatpotentially contain the first portion and the second portion from thesame polynucleotide based on the results from steps (b) and (c).
 3. Themethod of claim 2, further comprising performing steps (i)-(iii).
 4. Themethod of claim 2, wherein step (d) further comprises comparing thelocation of each of the plurality of nucleic acid clusters on thesurface, wherein the distance from one of the plurality of nucleic acidclusters to another of the plurality of nucleic acid clusters determinesthe probability of whether the sequence of the first portion of the oneof the plurality of double-stranded polynucleotide molecules and thesequence of the second portion of the one of the plurality ofdouble-stranded polynucleotide molecules are located on the same targetpolynucleotide molecule.
 5. The method of claim 2, wherein step (c) isperformed prior to step (b).
 6. The method of claim 2, wherein the knownlength is a known average length.
 7. The method of claim 2, wherein theknown length of each of the plurality of polynucleotides is within arange of sizes.
 8. The method of claim 2, wherein the first portion andsecond portion are noncontiguous.
 9. The method of claim 2, wherein thesurface is a patterned surface comprising a first population ofoligonucleotides and a second population of oligonucleotides.
 10. Themethod of claim 2, wherein step (c) is carried out by sequencing byligation, sequencing by synthesis or sequencing by hybridization. 11.The method of claim 2, further comprising comparing the sequences of thefirst portion and second portion to a reference sequence.
 12. The methodof claim 2, wherein each of the plurality of double-strandedpolynucleotide molecules comprises indexing tags.
 13. The method ofclaim 12, wherein the first portion and second portion of each of theplurality of double-stranded polynucleotide molecules comprise the sametag from the indexing tags.
 14. The method of claim 13, wherein thefirst portion and second portion of each of the plurality ofdouble-stranded polynucleotide molecules retain said same tag after step(ii).
 15. The method of claim 2, wherein the surface is a surface of aflow cell.
 16. The method of claim 2, wherein step (i) comprises:producing fragments by fragmenting a genomic DNA, adding adaptors to theends of the fragments, thereby obtaining the plurality ofdouble-stranded polynucleotides.